Ultrastructural Localization of Hydrogen Peroxide Production in Ligninolytic Phanerochaete chrysosporium Cells

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Abstract
Previous studies have shown that the hydroxyl radical derived from hydrogen peroxide (H2O2) is involved in lignin degradation by Phanerochaete chrysosporium. In the present study, the ultrastructural sites of H2O2 production in ligninolytic cells of P. chrysosporium were demonstrated by cytochemically staining cells with 3,3′-diaminobenzidine (DAB). Hydrogen peroxide production, as evidenced by the presence of oxidized DAB deposits, appeared to be localized in the periplasmic space of cells from ligninolytic cultures grown for 14 days in nitrogen-limited medium. When identical cells were treated with DAB in the presence of aminotriazole, periplasmic deposits of oxidized DAB were not observed, suggesting that the deposits resulted from the H2O2-dependent peroxidatic oxidation of DAB by catalase. Cells from cultures grown for 3 or 6 days in nitrogen-limited medium or for 14 days in nitrogen-sufficient medium had little ligninolytic activity and low specific activity for H2O2 production and did not contain periplasmic oxidized DAB deposits. The results suggest that in cultures grown in nitrogen-limited medium, there is a positive correlation between the occurrence of oxidized DAB deposits, the specific activity for H2O2 production in cell extracts, and ligninolytic activity. Images